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Evaluation of Toxicity and Mutagenicity of

Trichosetin, an Anti-MRSA Antibiotic Produced from

the Dual Culture of Catharanthus roseus Callus and

Trichoderma harzianum

Presentation of Completed Researches

UPLB DA-BAR RDE Network Headquarters, UPLB, College, Laguna. March 30, 2010

EUFROCINIO C. MARFORI, Ph.D.

University Researcher, BIOTECH, UPLB

Trichosetin

NHO

O

O

OH

(Marfori et al., 2002. Z. Naturforsch. 57c: 465-470)

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Diwa and Peji (2005) established the antimicrobial activityof trichosetin against local clinical MRSA isolates.

Vancomycin

Trichosetin

Methanol

Methicillin-resistant Staphylococcus aureus

(MRSA)

-minor skin infections

-life-threatening diseases

such as pneumonia,

meningitis, endocarditis,

toxic shock syndrome and

septicemia.

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OO

O

O

OCH2OH

HOHO

H2NHO

Cl

NH

O

HN

O

NH2

O

O

NH

OHN

HN

HO

O

HO

OHHOOH

O

O

Cl

Vancomycin

Recent emergence of vancomycin-intermediate resistant S.

aureus (VISA) is now raising serious public health concerns.

•Any new compound proven to be effective against

certain microbial pathogens should first undergo

assessment of the potential risk it may cause to the

user, i.e. mutagenicity and toxicity studies.

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Objectives

General Objective:

To evaluate the potential risk of trichosetin as a therapeutic agent for

methicillin- resistant Staphylococcus aureus (MRSA) infection

Specific objectives:

a. To establish and maintain the dual culture of Catharanthus roseus callus

and Trichoderma harzianum

b. To determine the oral LD50 of trichosetin in mice

c. To determine the effect of trichosetin on vital organs

d. To determine the mutagenicity of trichosetin

Establishment of the dual culture of

Catharanthus roseus callus and Trichoderma harzianum

C. roseus plant

C. roseus callus dual culture

Trichoderma harzianum

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Dual culture (10 kg)

MeOH extract (100.11 g)

hexane

layerAqueous

layer

EtOAC extractAqueous

(2.82 g)

Isolation of trichosetin

soaked in 10 L MeOH overnight

resuspended in 500 ml dH2O

adjusted to pH 4.0; extracted

with 500 ml n-hexane (3x)

extracted with 500

ml EtOAc (3x)

open- column chromatography

34.5 cm x 1.6 cm column

30 g deactivated silica

eluted with solvents of

increasing polarity

EtOAc fraction

Fraction No. 7- Methanol (145.2 mg)

Fraction No. 1- Toluene

Fraction No. 2- Toluene: Acetone (9:1)

Fraction No. 3- Toluene: Acetone (8:2)

Fraction No. 4- Toluene: Acetone (7:3)

Fraction No. 5- Toluene: Acetone (6:4)

Fraction No. 6- Acetone

rec Assay

• involves the use of two strains of

Bacillus subtilis :

recombination-proficient strain H17 Rec+

recombination-deficient strain M45 Rec-

rec Assay- used to determine DNA-damaging capacity of a compound

• M45 Rec- is much more sensitive toagents which cause genetic alterations

• a test compound which inhibited the growth of M45 Rec- may be

considered as DNA-damaging and potential mutagen.

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Dilution of trichosetin

Streaking of the test strainsPlacing the paper disks containing

trichosetin

Prepared plates for the rec assay

10 ppm

trichosetin

100 ppm

trichosetin

1000 ppm

trichosetinquinoline

(positive control)

methanol

(negative control)

TreatmentLength of inhibition, mm

M45 Rec-H17 Rec+

Trichosetin, 10 ppm 0 0

Trichosetin, 100 ppm 0 0

Trichosetin, 1000 ppm 0 0

Methanol 0 0

4-nitroquinoline oxide 23.1 + 1.2 31.2 + 1.6

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Ames Test

- to determine the ability of a compound to induce frameshift and point

mutation

- being done using special tester strains of S.

typhimurium (TA 98 and TA 100), which are

his- and cannot synthesize histidine so that

they are unable to grow in its absence.

- tester strains spread on a culture plate that

lacks histidine

- a mutagen placed in the culture medium caused some of these his- bacteria

to revert to the his+ phenotype, as detected by their growth into visible

colonies after 18 h at 37oC

- mutagenicity of a compound scored based on the number of induced

revertant colonies

- a compound is mutagenic if the number of the induced revertant colonies is

more than double the revertant colonies of the negative control

Ames test of trichosetin using Salmonella typhimurium TA 98 and TA 100

Treatment Ave. Microbial Count (CFU mL-1)

TA 98 TA 100

No substance (inoculated with bacteria only)a 5.00 108.67

Trichosetin, 10000 mg.L-1 2.33 35.00

Trichosetin, 1000 mg.L-1 3.33 73.00

Trichosetin, 100 mg.L-1 7.67 42.67

Trichosetin, 10 mg.L-1 5.66 108.33

Methanol 2.33 23.67

Acridine orange, 1000 mg.L-1 32.66 546.00

aPlate for revertant. A >2-fold reversion rate indicates that the substance is mutagenic.

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Micronucleus Test

Micronucleus test- for determination of the chromosome-damaging property of

a test compound.

DNA damage by a mutagen causes the formation of acentric chromatids and

chromosome fragments.

After mitosis, acentric chromatids and

chromosome fragments are included in the

daughter cells but a considerable

proportion is transformed into one or

several secondary nuclei which are smaller

than the principal nucleus and are called

micronuclei.

Intraperitoneal administration of

trichosetin Cervical dislocation of mouse

Flushing the bone marrow with

fetal calf serumBlood smearing

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Trichosetin, 10 mg/kg 3.53 + 1.93

Trichosetin, 50 mg/kg 3.87 + 1.02

Trichosetin, 100 mg/kg 3.00 + 1.15

Methanol (negative control) 2.07 + 1.62

Tetracycline (positive control) 7.07 + 2.62

Treatment No. of MPEs

Note: A >2-fold increase in the frequency of micronuclei indicates that the

substance is mutagenic.

Mean number of multinucleated polychromatic erythrocytes

(MPEs) scored per 1000 polychromatic erythrocytes (PCEs)

Toxicity Test:

Route: OralDose levels tested: 0, 0.16, 0.25, 0.40, 0.63, 0.80 g/Kg body wt.

Test animal: ICR-strain Swiss Webster mice

Parameters taken:

1. weight of mice

2. mortality/morbidity

3. gross morphological

anatomy of major internal

organs

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Mean body weight of male and female ICR-strain Swiss

Webster mice over the 14-d observation period

male female

Dose (g/Kg)

0 0 0 -

0.16 0 0 -

0.25 0 33 Ataxia, catalepsy

0.40 50 67

0.63 75 100

0.80 100 -

% Mortality Observed Toxidromes % Morbidity

Ataxia, catalepsy,

analgesia, dyspnea,

pilomotor erection,

fine body tremors

Ataxia, catalepsy,

analgesia, dyspnea,

pilomotor erection,

coarse body tremors,

cyanosis, loss of

screen grip,

anesthesia, convulsion

Lethal and non-lethal effects of trichosetin

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Gross anatomy of major internal organs from male

ICR-strain Swiss Webster mice

0 0.16 0.25 0.40 0.63

g /Kg

Gross anatomy of major internal organs from

female ICR-strain Swiss Webster mice

0 0.16 0.25 0.40 0.63

g /Kg

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Conclusion

Rec assay, Ames test and Micronucleus test showed that

trichosetin is not mutagenic.

Toxicity assay showed a dose-related increase in the

magnitude of biological response observed in the

toxidromes. Increasing doses results to decreasing body

weights; however, no dose-related changes were

observed in the gross morphological anatomy of major

internal organs.

LD 50 in mice= 0.40 g/ Kg; NOAEL= 0.16 g/Kg

Based on therapeutic index, it was found that

trichosetin has a wide margin of safety as an antibiotic

against MRSA.

Acknowledgements

UPLB Basic Research Program

Tonton Alad, Angeli Cocos, Bia Catibog, Lian Caluag and

Prof. Marilen Parungao Department of Biology, CAS, UP Manila

Danica Dimaya and Dr. Evangeline Amor

Institute of Chemistry, UP Diliman

Bureau of Animal Industry, Quezon City

Dr. Nelia Cortes-Maramba, Dr. Sid Sia and Prof. Susie Sio, Department

of Pharmacology, College of Medicine, UP Manila

Dr. Ernesto Balolong, Jr., NIH, UP Manila

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Related Publications:

1. Marfori, E.C., Kajiyama, S., Fukusaki, E. and Kobayashi, A. 2002. Trichosetin, a novel

tetramic acid antibiotic produced in the dual culture of Trichoderma harzianum and

Catharanthus roseus callus. Zeitscrift fur Naturforschung 57c: 465-470.

2. Marfori, E.C., Bamba, T., Kajiyama, S., Fukusaki, E. and Kobayashi, A. 2002.

Biosynthetic studies of the tetramic acid antibiotic trichosetin. Tetrahedron 58: 6655-

6658.

3. Marfori, E.C., Kajiyama, S., Fukusaki, E. and Kobayashi, A. 2003. Phytotoxicity of the

tetramic acid metabolite trichosetin. Phytochemistry 62: 715-721.

4. Marfori, E.C. and Malasa, A.B. 2007. Efficacy, mutagenicity and toxicity of trichosetin

as a new antibiotic for poultry. Phillipp. Agric. Scientist 90(2): 91-95.

5. Alad, P.O., Cocos, A.S., Balolong, E.C., Parungao, M.M. and Marfori, E.C. 2009.

Mutagenicity potential of the novel drug trichosetin estimated by using the rec assay

and micronucleus test. Philipp. J. Sci.138(2): 119-124.

6. Caluag, M.B., Catibog, I.D., Balolong, E.C., Parungao, M.M. , Cortes-Maramba, N.P.,

Sia, I.C., Sio, S.O. and Marfori, E.C. 2010. Toxicity of the novel drug trichosetin

(In Preparation)